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Image Processing Software
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Image J
Licence: FREE
Operating Systems: Windows, Mac OS, Linux
Key features: Multi-platform. Routine processing, analysis and measurement of multi-channel and colour 2D and 3D stacks, time-series
ImageJ is probably the most commonly used software for scientific image processing. It is free and runs on all platforms, which means anyone can run it anywhere. It has a set of basic image processing tools that will allow you to carry out such procedures as merging colour channels, editing stacks and making movies. You can threshold images and stacks and segment and measure features. You can then export the resulting data in a variety of formats (e.g TIFF, AVI, CSV). Much of the power of ImageJ comes through the plugins written by the ImageJ community. ImageJ has a built-in plugin language and editor if you know Java but if you want to create processing and analysis routines based on existing code it has a macro language and a macro recorder for linking together pre-existing tasks in a sequence. ImageJ is the basis of the Fiji analysis suite, μmanager device control software and OpenSPIM light sheet software
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ImageJ does not open all manufacturers' proprietary image file formats 'out of the box' but if you install the BioFormats Importer plugin this will read a huge and ever expanding range of formats. Specific analysis procedures tend to need specific plugins, which means one person's ImageJ installation can be very different to another's
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. This makes troubleshooting difficult, which is why I tend to remommend Fiji for most purposes. Although ImageJ opens and processes 3D and 4D data very well it's tools for 3D rendering and visualisation have not been as easy to use as those in other software when I have tried them.
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Fiji
Licence: FREE
Operating Systems: Windows, Mac OS, Linux
Key features: Multi-platform. Routine processing, analysis and measurement of multi-channel and colour 2D and 3D stacks, time-series. Updater, plugins include BioFormats Importer
Fiji is an extended version of ImageJ with two major advantages. Firstly, it has an updater that allows you to keep versions constant, making troubleshooting at lot easier. Secondly, it comes bundled with pre-installed plugins (including the aforementioned BioFormats Importer) so many analysis features are already in there and you don't need to install them yourself. If you do need to install plugins this is just as easy as it is for ImageJ. I currently recommend Fiji as the software that all staff members should use for basic image processing procedures.
Fiji comes bundled with plugins but these seem to be heavily weighted towards certain applications that aren't necessarily cell biological. This probably simply reflects who is writing software for it. It is therefore likely that from time to time you will need to instal a plugin. This may be fine but some plugins may have dependencies (e.g. Java 3D) that aren't necessarily installed with Fiji.
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Volocity
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Licence: 500 licences for the Volocity core program on the LMCB network. 3 licences each for the Restoration, Visualization and Quantitation modules
Operating Systems: Windows and Mac OS
Key features: 3D surface rendering, volume rendering and ray tracing. Identify and measure 3D objects and object properties. Deconvolution.
Volocity natively analyses data sets in 3D rather than as stacks of 2D slices
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. This means that measurements automatically refer to 3D objects rather than single slices and are given in 3D units (e.g. cubic microns). Volocity consists of a core programs that allows you to open your data, carry out some basic processing and export in a variety of formats. If you want to carry out advanced processing in Volocity you need to use one of the analysis modules: Restoration, Visualization and Quantitation. Restoration is a deconvolution package
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of most use with high quality confocal data. Visualization is a 3D reconstruction and rendering package with volume and surface rendering tools and a ray tracer. Quantitation is a measurement package that can be used to apply a 'pipeline' of analysis steps to process data and extract measurements. Basically this means thresholding and segmentation, binary style procedures like erosion and dilation, object identification, filtering by object features, measurement and colocalisation, etc.
I find that the Restoration module is not great at deconvolving data with lower signal to noise ratios, so I tend to use Huygens and AutoQuant for deconvolution.
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It is difficult to
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set up an analysis pipeline using the Quantitation but all such tools are difficult and require a lot of trial and error, so this isn't unusual. The analysis and charts tools in the Quantitation module are not intuitive at all so most of the time I export data as a spreadsheet for analysis in other packages (e.g. Excel). The Tracking tools for measuring objects moving over time seem to be a lot more flexible and user friendly in Imaris.
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How to get a copy of Volocity to use on your computer (LMCB network only)
Volocity Registration Correction
Volocity Acquisition Quick Start
Imaris
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Licence: Single licence installed on Beast analysis computer
Operating Systems: Windows (but can also run on Linux)
Key features: 3D surface and volume rendering. Identify and measure 3D objects and object properties.
Imaris is a 3D rendering and quantification software package. 3D rendering and identification of objects, spots and other features are done using the Surpass view, which will probably be the one you use the most. Imaris does not have a built-in deconvolution package (Bitplane use AutoQuant for that) but it does have the advantage that measurements can be made on the 3D rendered volume itself, which is something that isn't possible in Volocity. It is also possible to use MatLab and Fiji macros and plugins using ImarisXT. The version of Imaris we have also has a Filament Tracer module, which is designed for tracing neurons but that could also be used for branching blood vessels
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. We are running Imaris 7.7 and have the following modules: MeasurementPro, ImarisTracking, ImarisColoc, ImarisXT, FilamentTracer and ImarisVantage.
Bitplane Learning Zone (includes video tutorials)
Huygens
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Licence: Huygens Essential wide-field, confocal and spinning disc on WOPR computer. Huygens Professional confocal and STED on STED and Orac computers
Operating Systems: Windows (but can also run on Mac OS and Linux)
Key features: Deconvolution, but also has tools for 3D reconstruction and visualisation.
Scientific Volume Imaging's Huygens is a modular deconvolution software package. It has a core program that can be used to open, visualise and export data but additional modules must be installed to deconvolve wide-field, confocal, spinning disc and STED data. Multi-photon data can also be deconvolved but we don't have this module yet. We have Huygens on three machines but the licences vary, so you may not be able to deconvolve your data on all the machines (e.g. wide-field, multi-photon and spinning disc data can only be deconvolved on WOPR, confocal data can be deconvolved on all three machines). Additional modules for 3D rendering and image analysis are available but are not installed on our systems so Huygens is mostly used for deconvolution. Huygens is my preferred deconvolution package because it handles low signal-to-noise ratio images well, it is easy to edit parameters in the deconvolution routine and SVI provides a lot of information on their website. You have to register with SVI and get a login to access much of the content on the website.
Scientific Volume Imaging homepage
SVI's YouTube channel - video tutorials
Nikon NIS-Elements
Full NIS-Elements offline packages are available on Son of Beast and Deep Thought. The software is available for PC only but a free NIS-Elements Viewer for PC or Mac is available for download from Nikon's website
Analysis Computers
Beast
Processor: 2x Intel XEON CPU X5650 2.67 GHz, 6 cores, 12 logical processors
RAM: 96 GB
Software: Imaris 7.6.57, Volocity 6.3, Amira 5.4.1, LAS AF Lite 2.4.1
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Processor: 2x Intel XEON CPU X5650 2.67 GHz, 6 cores, 12 logical processors
RAM: 96 GB
Software: Volocity 6.3, Amira 5.4.2
Contact Jemima Burden or Ian White for training on Amira and Andrew Vaughan for training on Volocity. This machine is administered by the Electron Microscopy Facility and booking is through the MRCLMCB_EM Faces Scheduling System group.
WOPR
Processor: Intel Xeon X5460 3.16 GHz, 4 cores, 4 logical processors
RAM: 28 GB
Software: Huygens Essential 14.06.1p5, Volocity 6.3, LAS AF 2.6.0
Contact Andrew Vaughan for training. This machine is administered by the Light Microscopy Facility and booking is through the MRCLMCB_LSM Faces Scheduling System group.
Deep Thought
Processor: 2x Intel XEON CPU E5-2620, 2 GHz, 6 cores 12 logical partitions
RAM: 32 GB
Software: Autoquant X3 3.0.2, Metamorph 7.7.6, Volocity 6.3, LAS AF Lite 3.1.0
Contact Andrew Vaughan for training. This machine is administered by the Light Microscopy Facility and booking is through the MRCLMCB_LSM Faces Scheduling System group.
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RAM: 16 GB
Software: LAS AF 3.3.0, Huygens Professional 14.06.1p5 10 (confocal and STED licence)
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